摘要
为研究小反刍兽疫病毒H基因过表达后对病毒增殖的影响,根据G en Bank中小反刍兽疫病毒疫苗株Nigeria 75/1 H基因序列,设计1对引物扩增H基因全长编码区序列,并将其连接至真核表达载体p EGF P-C 3上,经鉴定正确后转染CHS-20细胞,并通过R T-PCR、荧光观察及W estern-blot分别在m RNA和蛋白水平检测H基因的表达。结果表明,H基因在CHS-20细胞内得到了表达。将p EGFP-C3-PPRV H重组质粒、空载体p EGFP-C3转染CHS-20细胞,待H基因表达后,接种PPRV Nigeria 75/1,病毒增殖一定时间后,以β-actin作为内参基因,采用相对荧光定量RT-PCR法研究病毒增殖量的变化。结果表明,相对于未转染及转染空载体p EGFP-C3的细胞,转染p EGFP-C3-PPRV H重组质粒的细胞PPRV增殖量得到了提高,且差异显著(P<0.05),这说明在CHS-20细胞中过表达PPRV H基因能促进PPRV增殖。
To study the effect on PPRV multiplication after overexpression of H gene,the whole cod- ing sequence of H gene of Peste des petits ruminants virus（PPRV）was amplified using RT-PCR and cloned into eukaryotic expression vector pEGFP-C3. Then pEGFP-C3-PPRV H plasmid was used to transfect the CHS-20 cells. In mRNA and protein levels,RT-PCR,fluorescence observation and Western-blot analysis were used to detect H gene expression. The results showed that the H protein was successfully expressed in CHS-20 cells. The CHS-20 cells were transfected with the recombinant plasmid and then infected with PPRVNigeria 75/i after 48 h. After 72 h,the virus titres were analysed by the relative quantitative RT-PCR. The results showed that the titre of PPRV in cells transfected with pEGFP-C3-PPRVH was higher than that in the untransfected cells and the cells transfected with pEGFP-C3 plasmid and there was sta- tistically significant difference（P〈0. 05,paired significantly promoted the PPRVmultiplieation. t-test）.The overexpression of PPRV H in CHS-20 cells
作者
赵玲娜
金红岩
薛晓娟
隋修锟
ZHENEY Makay
梁琳
李刚
ZHAO Ling-na JIN Hong-yan XUE Xiao-juan SUI Xiu-kun ZHENEY Makay LIANG Lin LI Gang(State Key Laboratory of A nimal NutritiordBeij＇ing Scientific Observation and Experiment Station for Veterinary Drug and Veterinary Biotechnology of the Ministry of Agriculture/Institute of A nimal Science and Veterinary Medicine, Chinese Academy of A gricutural Sciences ,Beijing 100193, China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2016年第11期1349-1357,共9页
Chinese Veterinary Science
基金
国家自然科学基金项目(31172342
31472203)
国家科技支撑计划(2013BAD12B05)
转基因重大专项(2014ZX08-01203B)
关键词
小反刍兽疫病毒
H基因
基因过表达
病毒增殖
peste des petits ruminants virus,H gene,gene overexpression,virus multiplication
